Rod-shaped plant virus nanoparticles as imaging agent platforms

ABSTRACT

A rod-shaped plant virus having an interior surface and an exterior surface, and at least one imaging agent that is linked to the interior and/or exterior surface is described. The rod-shaped viruses can be combined into larger spherical nanoparticles. A rod-shaped plant virus or spherical nanoparticles including an imaging agent can be used in a method of generating an image of a tissue region of a subject such as a tumor or atherosclerotic tissue by administering the virus particle to the subject and generating an image of the tissue region of the subject to which the virus particle has been distributed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/711,492, filed Oct. 9, 2012, which is incorporated herein byreference.

GOVERNMENT FUNDING

The present invention was supported by NIH/NIBIB grant P30 EB011317 (toNFS)), Mt. Sinai Foundation (to NSF), and NIH/NHLBI training grant T32HL105338. The Government has certain rights in this invention.

BACKGROUND

Magnetic resonance imaging (MRI) is an emerging technology used fordetection of disease and for follow-up diagnosis after surgery ortreatment. While MRI shows great potential because of its high spatialresolution, deep soft tissue contrast, and use of non-ionizingradiation, its low sensitivity remains a drawback. To overcome thisshortcoming, paramagnetic contrast agents, such as Magnevist® (an FDAapproved chelated gadolinium reagent), can be used to enhance thedetection sensitivity of MRI. The conjugation of contrast agents to amacromolecular platform further enhances imaging sensitivity. Reducedmolecular tumbling rates of gadolinium ions after conjugation to suchnanoparticles results in increased longitudinal relaxivities. Waters, E.A. & Wickline, S. A., Basic Research in Cardiology, 103, 114-121 (2008).Furthermore, multivalent display results in increased localconcentration, both events are contributing to increased sensitivity.Various nanoparticle systems have been explored as supramolecularcontrast agents; these include dendrimers, liposomes, perfluorocarbons,silica, as well as protein cages and virus-based nanoparticles, alsotermed viral nanoparticles (VNPs). Bruckman, M, Steinmetz, N,“Engineering Gd-loaded nanoparticles to enhance MRI sensitivity via T₁shortening,” Nanotechnology, 24, (46) (2013).

VNPs, specifically plant viruses and bacteriophages, have receivedtremendous attention in recent years. They have been developed asresearch tools and platforms for materials science as well as forpotential nanomedical applications. Lee et al., Biotechnol Bioeng. 109,16-30 (2012). The propensity to self-assemble around a cargo (thegenome) and to deliver this cargo to specific cells and tissues, makeviruses ideal candidates for site-specific delivery of therapeuticsand/or contrast agents. Indeed, several VNP-based technologies are inclinical testing for gene delivery and oncolytic virotherapy. VNPs areattractive materials because of their high degree of symmetry,polyvalency, monodispersity, and their genetic or chemicalprogrammability. Most VNP structures have been solved to atomicresolution, which allows tailoring with a high degree of spatialcontrol. Using chemoselective bioconjugation reactions, VNPs can bemodified with imaging contrast agents, therapeutic moieties, and/ortargeting ligands such as peptides or antibodies. For example,preclinical imaging of prostate tumors has been demonstrated usingcowpea mosaic virus (CPMV) modified with prostate cancer-specificpeptide ligands (bombesin) and near infrared imaging dyes. Steinmetz etal., Small 7, 1664-1672 (2011). Moving toward translational research,several research groups have engineering VNPs with paramagnetic MRIcontrast agents. Similar to other nanoparticles, increased relaxivitiesare achieved based on reduced tumbling rate of the contrast agent. Huanget al., Theranostics 2, 86-102 (2012). For example, bacteriophage MS2, a27 nm sphere, was loaded with ˜180 chelated Gd molecules using a TOPOligand and was able to achieve ionic relaxivities of up to 41.2 mM⁻¹s⁻¹per Gd ion and 7,416 mM⁻¹s⁻¹ per nanoparticle. In comparison, Magnevist®has a relaxivity 5.2 mM⁻¹s⁻¹. Anderson et al. Nano Letters 6, 1160-1164(2006); Garimella et al., JACS 133, 14704-14709 (2011).

To date, research and development of VNP-based MRI contrast agents hasfocused on spherical platforms; however, this may not be optimal. Recentwork by the inventors and others indicates improved pharmacokinetics,increased immune evasion (e.g., reduced macrophage uptake), increasedtumor homing, tissue penetration, and vessel wall targeting of elongatedparticles, e.g. potato virus X and tobacco mosaic virus Shukla et al.Molecular pharmaceutics, 10(1):33-42 (2013), Lee et al. BiomaterialsScience 1, 581-588 (2013). Wen et al. Biological Physics,39(2):301-25(2013). However, the use of rod-shaped virus particles asimaging agents remains unexplored.

SUMMARY

The inventors have explored the use of tobacco mosaic virus (TMV) as ascaffold for multivalent display of paramagnetic MRI contrast agents.TMV is a rod-shaped VNP measuring 300×18 nm with a solvent-accessible 4nm-wide interior channel What makes TMV particularly interesting is therecent discovery that TMV can undergo thermal transition to formRNA-free spherical nanoparticles (SNPs). Atabekov et al., Journal ofGeneral Virology 92, 453-456 (2011). The size of SNPs can be tightlytuned through adjustment of TMV concentration with sizes ranging from100-300 nm (0.1-1.0 mg/ml) to 300-800 nm (1-10 mg/ml). TMV thereforeprovides a unique platform to study rod-shaped and sphericalnanomaterials side-by-side.

Each TMV nanorod is formed from 2130 copies of an identical coat proteinthat is helically arranged around a single strand RNA. Klug, A., PhilosTrans R Soc Lond B Biol Sci 354, 531-535 (1999). The rigid structure ofTMV displays 2.2 times more coat proteins per cubic nanometer than itsspherical (˜30 nm diameter) VNP counterparts, thus allowing moreefficient loading of cargos (contrast agents, therapeutics, and ortargeting ligands). Engineering TMV particles has yielded a variety ofmaterials for tissue engineering scaffolds, vaccine development, and awide array of electronic materials. However, they have not previouslybeen used as a drug delivery vehicle or contrast agent.

The inventors describe herein the formulation of a novel class of MRIcontrast agents based on TMV nanorods and spheres. They show thatcontrast agent-loaded rod-shaped TMV can undergo thermal transition toform a spherical contrast agent. Conjugation of rod-shaped TMV withDOTA-Gd at either the exterior surface or interior channel was achievedusing a combination of amide coupling, diazonium chemistry, andCu(I)-catalyzed azide alkyne cycloaddition reactions. Particlemodification and stability is confirmed with MALDI-TOF mass spectroscopy(MS), inductively coupled plasma optical emission spectrometry(ICP-OES), denaturing gel electrophoresis (SDS-PAGE), size exclusionchromatography (SEC), and transmission and scanning electron microscopy(TEM and SEM). Thermal re-shaping was then applied to generatehigh-relaxivity nanospheres. TMV rods with relaxivities up to ˜35,000mM⁻¹s⁻¹ and TMV SNPs with relaxivities of close to 400,000 mM⁻¹s⁻¹ weregenerated (measured at 60 MHz); these formulations display the highestrelaxivities reported to date using VNP scaffolds. Finally, MR phantomsof varying concentrations were imaged using a pre-clinical 7.0 T and aclinical 1.5 T MRI.

In addition, a rod-shaped tobacco mosaic virus was used to target andimage atherosclerotic plaques in vivo. TMV was loaded with magneticresonance and fluorescence contrast agents to provide a dual-modalimaging platform. Targeting to atherosclerotic plaques was achieved withvascular cell adhesion molecule (VCAM) receptors present on activatedendothelial cells. Dual, molecular imaging was confirmed using a mousemodel of atherosclerosis.

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more readily understood by reference to thefollowing drawings.

FIG. 1 provides the chemical structures of several common gadolinium(Gd) chelating molecules.

FIG. 2 provides a PyMol image of tobacco mosaic virus highlighting theinterior glutamic acids, GLU97 and GLU106, exterior tyrosine, TYR139,for bioconjugation. Additional glutamic and aspartic acid residues andtyrosine residues are highlighted for reference.

FIG. 3 provides (A) schematic illustration of the bioconjugationreactions used to incorporate terminal alkynes to the interior andexterior of TMV; (B) schematic illustration of the CuAAC reaction tolabel TMV particles with Gd(DOTA); and MALDI-TOF MS of (C) eGd-TMV and(D) iGd-TMV. In the MALDI-TOF MS, peaks labeled with eAlk and iAlk referto the alkyne labeled proteins, 1-Gd, 2-Gd, and 3-Gd refer to coatproteins labeled with one, two, and three Gd(DOTA).

FIG. 4 provides (A) schematic illustration of the thermal transitionfrom rod-shaped TMV to spherical nanoparticles; representative TEMimages of (B) iGd-TMV, (C) eGd-TMV to SNP, (D) iGd-TMV to SNP, 10seconds, and (E) iGd-TMV to SNP 15 seconds; and (F) SEM image ofiGd-SNPs with (G) the corresponding DLS (Nanosight size analyzer). Scalebars=500 nm.

FIG. 5 provides (A) phantom images of tubes containing eGd-TMV, iGd-TMVand Gd(DOTA) with the corresponding Gd concentrations in μM; measuredusing a clinical 1.5 T MRI. Plot of 1/T₁ versus Gd concentration (mM)for eGd-TMV (B) and iGd-TMV (C) taken from three MR sources. The slopesof the plots correspond to the ionic relaxivity. Data were collected atvarying field strengths (300 MHz, 64 MHz, and 60 MHz).

FIG. 6 provides a graph comparing the nanoparticle relaxivities (leftaxis) and ionic relaxivities (right axis) of TMV particles described inExample 1 against other Gd-VNPs at 60 MHz (refs 1, 2, or 5) or 64 MHz(refs 3, 4, and 6). The cited references are: #1 Garimella et al., JACS,133, 14704-14709 (2011); #2 Hooker et al., Nano Letters 7, 2207-2210(2007); #3 Liepold et al., Magn Reson Med 58, 871-879 (2007); #4Anderson et al., Nano Letters 6, 1160-1164 (2006); #5 Pokorski et al.,JACS 133, 9242-9245 (2011); #6 Prasuhn et al., Chemical Communications28, 1269-71 (2007).

FIG. 7 provides a reaction scheme showing the bioconjugation sequenceused to engineer TMV particles and data table showing labelingefficiencies of TMV with Cy5 optical dye, Gd(DOTA) MR contrast agent,the T1 relaxivity at 60 MHz in mM⁻¹s⁻¹, and the number of VCAM(targeting ligand, also includes PEG) and PEG only (negative control)per TMV.

FIG. 8 provides graphs and images based on characterization of VCAM-TMVand PEG-TMV by TEM imaging after UAc staining (A, B), size exclusionchromatography (C, D), SDS-PAGE (E) and MALDI-TOF mass spectra (F).SDS-PAGE lane assignments are: 1-Wt-TMV, 2-eViA-TMV, 3-ePiA-TMV,4-VCAM-TMV (after labeling with Cy5 and Gd(DOTA)), 5-PEG-TMV (afterlabeling with Cy5 and Gd(DOTA)).

FIG. 9 provides ex vivo optical imaging of aortas from ApoE^(−/−) miceafter administration of targeted TMV sensors and respective control anda bar graph showing pixel intensity mean after quantitative imageanalysis using Maestro Imager software.

FIG. 10 provides images showing pre- and post-injection MRI scans of (A)VCAM-TMV, (B) Gd(DOTA), and (C) PBS. The third column is the subtractedimage. Insets are magnified images of the abdominal aorta region ofinterest.

DETAILED DESCRIPTION

Rod-shaped plant viruses including at least one imaging agent that islinked to the interior and/or exterior surface of the virus aredescribed. The rod-shaped viruses can be combined into sphericalnanoparticles. The rod-shaped plant virus particles or sphericalnanoparticles including an imaging agent can be used in a method ofgenerating an image of a tissue region of a subject such as a tumor oratherosclerotic tissue by administering the virus particle to thesubject and generating an image of the tissue region of the subject towhich the virus particle has been distributed.

Definitions

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. As used in this specificationand the appended claims, the singular forms “a”, “an” and “the” includeplural references unless the content clearly dictates otherwise. Thus,for example, reference to “a cell” includes a combination of two or morecells, and the like.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or 110%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

“Image” or “imaging” refers to a procedure that produces a picture of anarea of the body, for example, organs, bones, tissues, or blood.

A “subject,” as used herein, can be any animal, and may also be referredto as the patient. Preferably the subject is a vertebrate animal, andmore preferably the subject is a mammal, such as a domesticated farmanimal (e.g., cow, horse, pig) or pet (e.g., dog, cat). In someembodiments, the subject is a human.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject for the methodsdescribed herein, without unduly deleterious side effects.

As used herein, the term “relaxation time” refers to the time requiredfor a nucleus which has undergone a transition into a higher energystate to return to the energy state from which it was initially excited.Regarding bulk phenomena, the term “relaxation time” refers to the timerequired for a sample of nuclei, the Boltzmann distribution of which hasbeen perturbed by the application of energy, to reestablish theBoltzmann distribution. The relaxation times are commonly denoted T₁ andT₂. T₁ is referred to as the longitudinal relaxation time and T₂ isreferred to as the transverse relaxation time. As used herein, the term“relaxation time” refers to the above-described relaxation times eithertogether or in the alternative. An exhaustive treatise on nuclearrelaxation is available in Banci, L, et al. Nuclear and ElectronRelaxation, VCH, Weinheim, 1991, which is herein incorporated byreference.

As used herein, the term “diagnostically effective amount” refers to anamount of contrast agent that is sufficient to enable imaging of thecontrast agent in cells, tissues, or organisms using imaging equipment.

In one aspect, the present invention provides a rod-shaped plant virushaving an interior surface and an exterior surface, and at least oneimaging agent that is linked to the interior and/or exterior surface ofthe virus.

Rod-Shaped Plant Viruses

A rod-shaped plant virus is a virus that primarily infects plants, isnon-enveloped, and is shaped as a rigid helical rod with a helicalsymmetry. Rod shaped viruses also include a central canal. Rod-shapedplant virus particles are distinguished from filamentous plant virusparticles as a result of being inflexible, shorter, and thicker indiameter. For example, Virgaviridae have a length of about 200 to about400 nm, and a diameter of about 15-25 nm. Virgaviridae have othercharacteristics, such as having a single-stranded RNA positive sensegenome with a 3′-tRNA like structure and no polyA tail, and coatproteins of 19-24 kilodaltons.

In some embodiments, the rod-shaped plant virus belongs to a specificvirus family, genus, or species. For example, in some embodiments, therod-shaped plant virus belongs to the Virgaviridae family. TheVirgaviridae family includes the genus Furovirus, Hordevirus,Pecluvirus, Pomovirus, Tobamovirus, and Tobravirus. In some embodiments,the rod-shaped plant virus belongs to the genus Tobamovirus. In furtherembodiments, the rod-shaped plant virus belongs to the tobacco mosaicvirus species. The tobacco mosaic virus has a capsid made from 2130molecules of coat protein and one molecule of genomic single strand RNA6400 bases long. The coat protein self-assembles into the rod likehelical structure (16.3 proteins per helix turn) around the RNA whichforms a hairpin loop structure. The protein monomer consists of 158amino acids which are assembled into four main alpha-helices, which arejoined by a prominent loop proximal to the axis of the virion. Virionsare ˜300 nm in length and ˜18 nm in diameter. Negatively stainedelectron microphotographs show a distinct inner channel of ˜4 nm.

In further embodiments, the rod-shaped plant virus particle can becombined with other rod-shaped plant virus particles by means of athermal transition to form an RNA-free spherical nanoparticle (SNP),also referred to herein as a spherical nanoparticle imaging platform.FIG. 4 provides a schematic illustration of the thermal transition fromrod-shaped virus particles to spherical nanoparticles. A sphericalnanoparticle imaging platform is a spherical arrangement of the coatproteins of a plurality of rod-shaped plant virus particles linked to animaging agent on an interior surface of the virus particle, formed bythermal transition of the rod-shaped virus particles. The SNPs can beformed from rod-shaped plant virus particles bearing imaging agentslinked to the interior surface of the rod-shaped plant virus particles.SNPs can be labeled with suitable chemicals prior or post thermaltransition; for example, NHS-based chemistries allow one to conjugatefunctional molecules to SNPs post thermal transition; the SNPs arestable and remain structurally sound after chemical modification. TheSNPs including imaging agent can be formed from rod-shaped plant virusparticles (e.g., TMV virus particles) by briefly heating the rod-shapedplant virus particles labeled with imaging agent on an interior surfaceof the virus particle. For example, the rod-shaped plant virus particlescan be induced to undergo a thermal transition into SNPs by heating atabout 96° C. for about 10 to about 20 seconds. Examples of suitablerod-shaped virus particles include Virgaviridae virus particles andtobacco mosaic virus particles. Any of the imaging agents describedherein can be used with the spherical nanoparticles. In someembodiments, the imaging agent is a chelated lanthanide such asgadolinium.

The SNPs are formed from the coat proteins of one or more individualrod-shaped plant virus particles. In various embodiments, the SNP can beformed from about 1 to 10 virus particles, from about 10 to about 20virus particles, from about 20 to about 30 virus particles, from about30 to about 40 virus particles, or from about 40 to about 50 virusparticles. Depending on the nature of the coat proteins, the number ofvirus particles incorporated, and the virus particle concentration inthe solution in which the thermal transition occurs, the sphericalnanoparticles can also vary in size. In some embodiments, the SNPs havea size from about 50 nm to about 800 nm. In further embodiments, theSNPs have a size from about 100 to about 300 nm, or from about 150 toabout 200 nm.

Spherical nanoparticles including imaging agents such as chelatedgadolinium provide several advantages. First, SNPs can include a highper-particle concentration of imaging agent. For example, SNPs caninclude from about 3,000 to about 30,000 imaging agents per sphericalnanoparticle, with about 20,000 to about 30,000 imaging agent moleculesper spherical nanoparticle in some embodiments. In addition, for MRIimaging agents such as chelated lanthanides, the SNPs including imagingagents can also exhibit very high relaxivity per particle. For example,SNPs including lanthanide imaging agents can exhibit a T₁ relaxivity perparticle from about 10,000 mM⁻¹s⁻¹ to about 500,000 mM⁻¹s⁻¹ at 60 MHz,with about 350,000 mM⁻¹s⁻¹ to about 450,000 mM⁻¹s⁻¹ at 60 MHz in someembodiments. Finally, SNPs are more rapidly cleared from the body, whichcan be advantageous with imaging agents that may have increased adverseside effects when they persist within the subject after imaging.

Imaging Agents

The rod-shaped plant virus particle is modified to carry an imagingagent; i.e., the plant virus carrier comprises an imaging agent.Examples of imaging agents include fluorescent compounds, radioactiveisotopes, and MRI contrast agents. For example, in some embodiments, theimaging agent is a fluorescent molecule for fluorescent imaging. Thedetectable group can be any material having a detectable physical orchemical property. Such imaging agents have been well-developed in thefield of fluorescent imaging, magnetic resonance imaging, positiveemission tomography, or immunoassays and, in general, most any imagingagent useful in such methods can be applied to the present invention.Thus, an imaging agent is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful imaging agents in the present invention includemagnetic beads (e.g. Dynabeads™) fluorescent dyes (e.g., fluoresceinisothiocyanate, AlexaFluor555, Texas red, rhodamine, and the like),radiolabels (e.g., ³H ¹⁴C ³⁵S, ¹²⁵I, ¹²¹I, ¹¹²In, ⁹⁹mTc), other imagingagents such as microbubbles (for ultrasound imaging), ¹⁸F, ¹¹C, ¹⁵O,(for Positron emission tomography), ⁹⁹mTC, ¹¹¹In (for single photonemission tomography), and chelated lanthanides such as terbium,gadoliniuum, and europium (e.g., chelated gadolinium) or iron (formagnetic resonance imaging). The choice of imaging agent depending onsensitivity required, ease of conjugation with the compound, stabilityrequirements, available instrumentation, and disposal provisions.

In some embodiments, the imaging agent is a magnetic resonance imagingagent. Disease detection using MRI is often difficult because areas ofdisease have similar signal intensity compared to surrounding healthytissue. In the case of magnetic resonance imaging, the imaging agent canalso be referred to as a contrast agent. Lanthanide elements are knownto be useful as contrast agents. The lanthanide chemical elementscomprises the fifteen metallic chemical elements with atomic numbers 57through 71, and include lanthanum, cerium, praseodymium, neodymium,promethium, samarium, europium, gadolinium, terbium, dysprosium,holmium, erbium, thulium, ytterbium, and lutetium. Preferred lanthanidesinclude europium, gadolinium, and terbium. In order to more readilyhandle these rare earth metals, the lanthanides are preferably chelated.In some embodiments, the lanthanide selected for use as a contrast agentis gadolinium, or more specifically gadolinium (III).

Contrast agents are used to enhance the differentiation between tissueregions in order to better image the tissue. The ionic relaxivity rateof a contrast agent describes its capacity for contrast enhancement. Therelaxivity rate can be affected by a number of factors, including theuse of a chelating agent. Unless indicated otherwise, all relaxivitymeasurements described herein are at 60 MHz, which is the field strengthat which the relaxivity was typically measured. A clinical 3.0 Teslamagnet measures at that field strength. However, it should be noted thatpreclinical imaging is often done at higher magnetic field strength, andthat the relaxivity can change with the field strength. The relaxivityrate per rod-shaped plant virus particle can also be increased byincreasing the number of agent molecules that are linked to the virusparticle. Rod-shaped plant virus particles of the invention that havebeen chemically modified to include contrast agents can exhibitrelaxivity rates from about 10,000 to about 40,000 mM⁻¹S⁻¹. In someembodiments, the virus particles bearing contrast agents exhibit T₁relaxivity rates of at least about 10,000 mM⁻¹S⁻¹, about 20,000 mM⁻¹S⁻¹,about 25,000 mM⁻¹S⁻¹, about 30,000 mM⁻¹S⁻¹, about 35,000 mM⁻¹S⁻¹, andabout 40,000 mM⁻¹S⁻¹ at 60 MHz.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the virusparticle. The ligand then binds to an anti-ligand (e.g., streptavidin)molecule which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands.

Conjugation of Imaging Agents

The invention makes use of a rod-shaped plant virus particle that hasbeen modified to carry an imaging agent. Including an imaging agentallows the virus particle to serve as a platform for the imaging agent.A rod-shaped plant virus (i.e., rod-shaped plant virus particle) thathas been modified to include an imaging agent is also referred to hereinas a rod-shaped plant virus carrier.

In general, imaging agents can be conjugated to the rod-shaped plantvirus by any suitable technique, with appropriate consideration of theneed for pharmacokinetic stability and reduced overall toxicity to thepatient. The term “conjugating” when made in reference to an agent and arod-shaped plant virus particle as used herein means covalently linkingthe agent to the virus subject to the single limitation that the natureand size of the agent and the site at which it is covalently linked tothe virus particle do not interfere with the biodistribution of themodified virus. The imaging agent can be linked to the interior or theexterior of the virus, while in some embodiments the imaging agent islinked to both the interior and the exterior of the virus. The locationof the imaging agent on the interior or exterior is governed by theamino acids of the viral coat protein that are selected as targetlinking sites.

An imaging agent can be coupled to a rod-shaped plant virus particleeither directly or indirectly (e.g. via a linker group). In someembodiments, the agent is directly attached to a functional groupcapable of reacting with the agent. For example, viral coat proteinsinclude lysines that have a free amino group that can be capable ofreacting with a carbonyl-containing group, such as an anhydride or anacid halide, or with an alkyl group containing a good leaving group(e.g., a halide). Viral coat proteins also contain glutamic and asparticacids. The carboxylate groups of these amino acids also presentattractive targets for functionalization using carbodiimide activatedlinker molecules; cysteines can also be present which facilitatechemical coupling via thiol-selective chemistry (e.g.,maleimide-activated compounds). Further, viral coat proteins containtyrosines, which can be modified using diazonium coupling reactions. Inaddition, genetic modification can be applied to introduce any desiredfunctional residue, including non-natural amino acids, e.g. alkyne- orazide-functional groups. See Hermanson, G. T. Bioconjugation Techniques.(Academic Press, 2008) and Pokorski, J. K. and N. F. Steinmetz, MolPharm 8(1): 29-43 (2011), the disclosures of which are incorporatedherein by reference.

Alternatively, a suitable chemical linker group can be used. A linkergroup can serve to increase the chemical reactivity of a substituent oneither the agent or the virus particle, and thus increase the couplingefficiency. Suitable linkage chemistries include maleimidyl linkers,which can be used to link to thiol groups, isothiocyanate andsuccinimidyl (e.g., N-hydroxysuccinimidyl (NHS)) linkers, which can linkto free amine groups, diazonium which can be used to link to phenol, andamines, which can be used to link with free acids such as carboxylategroups using carbodiimide activation. Useful functional groups arepresent on viral coat proteins based on the particular amino acidspresent, and additional groups can be designed into recombinant viralcoat proteins. It will be evident to those skilled in the art that avariety of bifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), can be employed as a linker group.Coupling can be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues.

Other types of linking chemistries are also available. For example,methods for conjugating polysaccharides to peptides are exemplified by,but not limited to coupling via alpha- or epsilon-amino groups toNaIO₄-activated oligosaccharide (Bocher et al., J. Immunol. Methods 27,191-202 (1997)), using squaric acid diester(1,2-diethoxycyclobutene-3,4-dione) as a coupling reagent (Tietze et al.Bioconjug Chem. 2:148-153 (1991)), coupling via a peptide linker whereinthe polysaccharide has a reducing terminal and is free of carboxylgroups (U.S. Pat. No. 5,342,770), and coupling with a synthetic peptidecarrier derived from human heat shock protein hsp65 (U.S. Pat. No.5,736,146). Further methods for conjugating polysaccharides, proteins,and lipids to plant virus peptides are described by U.S. Pat. No.7,666,624.

When attaching lanthanide imaging agents such as gadolinium ions achelating compound is also used. Conjugation of a chelated lanthanideion to a virus particle can decrease its molecular tumbling rate,resulting in an increased ionic relaxivity rate. A number of chelatingcompounds have been developed to increase the coordinated watermolecules for lanthanide ions, which can almost double the relaxivityrate. Examples of effective gadolinium chelating molecules include1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),diethylenetriaminopentacetate (DTPA),1,4,7,10-tetraazacyclododecane-1,4,7-triasacetic acid (DOTA),6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid (AAZTA), and4-carboxyamido-3,2-hydroxypyridinone (HOPA), which are shown in FIG. 1 .See Gugliotta et al., Org. Biomol. Chem., 8, 4569 (2010), the disclosureof which is incorporated herein by reference. Bifunctional chelatingagents including N-hydroxysuccinimide/isothiocyanates, amine, maleimide,and azide chemical linkers can be used for conjugation to amines,carboxylatic acids, thiols, and alkynes.

In some embodiments, more than one type of imaging agent can be attachedto a rod-shaped plant virus particle. For example, a rod-shaped virusparticle can be made useful as an imaging agent for two or moredifferent visualization techniques. In further embodiments, differencesin the linking sites available on the outside surface (i.e., exterior)and inside channel (i.e., interior) of the virus particle can be used toprovide a virus particle with different imaging agents on the inside andoutside of the virus particle. For example, the virus particle can havea first imaging agent on the inside of the particle, and a second,different imaging agent on the outside of the virus particle. Thedifferent linking sites allow different linking chemistries to be usedfor the interior and exterior portions of the virus particle. Forexample, as shown in FIG. 3 , diazonium chemistry can be used to attachagents to phenol groups that are accessible on the exterior of a virusparticle, while amine chemistry is used to attach agents to carboxylgroups present on the interior of the virus particle. In furtherembodiments, rather than including a different imaging agent, differentlinking sites can be used to attach a therapeutic agent and/or atargeting moiety to the virus particle.

One advantage of the rod-shaped virus particles of the present inventionis that they have a higher number of coat proteins per cubic nanometeras compared with other viral particles, allowing more efficient loadingof imaging agents onto the virus particle. The number of imaging agentsthat can be loaded onto the virus particle depends on the number ofattachment sites available and the chemistries employed to link theagents to the virus particle. In some embodiments, each virus particleis loaded with about 500 agent molecules. In further embodiments, eachvirus particle is loaded with at least about 1,000, 1,500, 2,000, 2,500,3,000, 3,500, 4,000, 4,500, or at least about 5,000 imaging agentmolecules.

Targeting Moieties

In some embodiments, a targeting moiety can also be attached to therod-shaped plant virus particle. By “targeting moiety” herein is meant afunctional group which serves to target or direct the virus particle toa particular location, cell type, diseased tissue, or association. Ingeneral, the targeting moiety is directed against a target molecule.Thus, for example, antibodies, cell surface receptor ligands andhormones, lipids, sugars and dextrans, alcohols, bile acids, fattyacids, amino acids, peptides and nucleic acids may all be attached tolocalize or target the virus particle to a particular site. In someembodiments, the targeting moiety allows targeting of the rod-shapedplant virus particles of the invention to a particular tissue or thesurface of a cell. Preferably, the targeting moiety is linked to theexterior surface of the virus to provide easier access to the targetmolecule.

In some embodiments, the targeting moiety is a peptide. For example,chemotactic peptides have been used to image tissue injury andinflammation, particularly by bacterial infection; see WO 97/14443,hereby expressly incorporated by reference in its entirety. Anotherexample, are peptides specific to fibrin or vascular cell adhesionmolecules to direct the imaging probe to sites of inflammation, such asan atherosclerotic plaque. In other embodiments, the targeting moiety isan antibody. The term “antibody” includes antibody fragments, as areknown in the art, including Fab Fab₂, single chain antibodies (Fv forexample), chimeric antibodies, etc., either produced by the modificationof whole antibodies or those synthesized de novo using recombinant DNAtechnologies. In further embodiments, the antibody targeting moieties ofthe invention are humanized antibodies or human antibodies. Humanizedforms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.

In some embodiments, the antibody is directed against a cell-surfacemarker on a cancer cell; that is, the target molecule is a cell surfacemolecule. As is known in the art, there are a wide variety of antibodiesknown to be differentially expressed on tumor cells, including, but notlimited to, HER2. Examples of physiologically relevant carbohydrates maybe used as cell-surface markers include, but are not limited to,antibodies against markers for breast cancer (CA 15-3, CA 549, CA27.29), mucin-like carcinoma associated antigen (MCA), ovarian cancer(CA125), pancreatic cancer (DE-PAN-2), and colorectal and pancreaticcancer (CA 19, CA 50, CA242).

In some embodiments, the targeting moiety is all or a portion (e.g. abinding portion) of a ligand for a cell surface receptor. Suitableligands include, but are not limited to, all or a functional portion ofthe ligands that bind to a cell surface receptor selected from the groupconsisting of insulin receptor (insulin), insulin-like growth factorreceptor (including both IGF-1 and IGF-2), growth hormone receptor,glucose transporters (particularly GLUT 4 receptor), transferrinreceptor (transferrin), epidermal growth factor receptor (EGF), lowdensity lipoprotein receptor, high density lipoprotein receptor, leptinreceptor, estrogen receptor (estrogen); interleukin receptors includingIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12,IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, VEGFreceptor (VEGF), PDGF receptor (PDGF), transforming growth factorreceptor (including TGF-α and TGF-β), EPO receptor (EPO), TPO receptor(TPO), ciliary neurotrophic factor receptor, prolactin receptor, andT-cell receptors. Receptor ligands include ligands that bind toreceptors such as cell surface receptors, which include hormones,lipids, proteins, glycoproteins, signal transducers, growth factors,cytokines, and others.

In some embodiments, the rod-shaped plant virus is used to target tissuein a subject without the use of a targeting moiety based on the abilityof rod-shaped plant virus particles to preferentially accumulate incertain tissues. In particular, the rod-shaped plant virus particleshave been shown to preferentially accumulate in diseased tissue, such ascancer tissue or inflamed tissue (e.g., atherosclerotic blood vessels).While not intending to be bound by theory, it appears that rod-shapedplant virus particles are taken up by blood components such asmacrophage cells of the immune system, which subsequently accumulate indiseased tissue (e.g., a tumor or atherosclerotic blood vessel), therebydelivering the rod-shaped plant virus to cells at the disease site.

A tumor is an abnormal mass of tissue as a result of abnormal growth ordivision of cells caused by cancer. Tumors can occur in a variety ofdifferent types of tissue such as the breast, lung, brain, liver kidney,colon, and prostate, can be malignant or benign, and generally vary insize from about 1 cm to about 5 cm.

Magnetic resonance angiography (MRA) is a type of MRI that generatespictures of blood vessels (e.g., arteries) to evaluate them for stenosis(abnormal narrowing) or aneurysms (vessel wall dilatations, at risk ofrupture). MRA can be used to evaluate the arteries of the neck andbrain, the thoracic and abdominal aorta, the renal arteries, and thelegs. Rod-shaped plant virus particles (or SNPs) including linkedimaging agents can be used to facilitate conducing MRA of blood vesselsfor various uses, including evaluation of the possible development ofatherosclerosis. Atherosclerosis is a chronic inflammatory response inthe walls of arteries, caused largely by the accumulation of macrophagesand white blood cells and promoted by low-density lipoproteins (LDL,plasma proteins that carry cholesterol and triglycerides) withoutadequate removal of fats and cholesterol from the macrophages byfunctional high-density lipoproteins (HDL). It is commonly referred toas a hardening or furring of the arteries, and is caused by theformation of multiple plaques within the arteries, which can be detectedby MRA.

Imaging a Tissue Region

In one aspect, the present invention provides a method of generating animage of a tissue region of a subject, by administering to the subject adiagnostically effective amount of a rod-shaped plant virus particlehaving an imaging agent linked to an interior and/or exterior surface ofthe virus particle, and generating an image of the tissue region of thesubject to which the rod-shaped plant virus particle has distributed. Inorder to generate an image of the tissue region, it is necessary for aneffective amount of imaging agent to reach the tissue region ofinterest, but it is not necessary that the imaging agent be localized inthis region alone. However, in some embodiments, the virus particlesbearing imaging agents are targeted or administered locally such thatthey are present primarily in the tissue region of interest. In someembodiments, SNPs formed from rod-shaped plant virus particles are usedinstead of rod-shaped plant virus particles. Examples of images includetwo-dimensional cross-sectional views and three dimensional images. Insome embodiments, a computer is used to analyze the data generated bythe imaging agents in order to generate a visual image. The rod-shapedplant virus particles can include any of the virus particles describedherein, such as Virgaviridae virus particles and tobacco mosaic virusparticles. The tissue region can be an organ of a subject such as theheart, lungs, or blood vessels. In other embodiments, the tissue regioncan be diseased tissue, or tissue that is suspected of being diseased,such as a tumor or atherosclerotic tissue. Examples of imaging methodsinclude fluoroscopy, computed tomography, positive emission tomography,and magnetic resonance imaging.

Means of detecting labels in order to generate an image are well knownto those of skill in the art. Thus, for example, where the label is aradioactive label, means for detection include a scintillation counteror photographic film as in autoradiography. Where the label is afluorescent label, it may be detected by exciting the fluorochrome withthe appropriate wavelength of light and detecting the resultingfluorescence. The fluorescence may be detected visually, by means ofphotographic film, by the use of electronic detectors such as chargecoupled devices (CCDs) or photomultipliers and the like. Finally simplecolorimetric labels may be detected simply by observing the colorassociated with the label.

“Computed tomography (CT)” refers to a diagnostic imaging tool thatcomputes multiple x-ray cross sections to produce a cross-sectional viewof the vascular system, organs, bones, and tissues. “Positive emissionstomography (PET)” refers to a diagnostic imaging tool in which thepatient receives a radioactive isotopes by injection or ingestion whichthen computes multiple x-ray cross sections to produce a cross-sectionalview of the vascular system, organs, bones, and tissues to image theradioactive tracer. These radioactive isotopes are bound to compounds ordrugs that are injected into the body and enable study of the physiologyof normal and abnormal tissues. “Magnetic resonance imaging (MRI)”refers to a diagnostic imaging tool using magnetic fields and radiowavesto produce a cross-sectional view of the body including the vascularsystem, organs, bones, and tissues. Suitable imaging agents should beused that will help generate an image of a tissue region in the contextof the imaging technique being used. For example, when using magneticresonance imaging, a suitable imaging agent is a chelated lanthanide.

In some embodiments, the viral imaging agents (rod-shaped and SNP) ofthe present invention are used for MRI. MRI provides a good contrastbetween the different soft tissues of the body, which makes itespecially useful in imaging the brain, muscles, the heart, and cancerscompared with other medical imaging techniques such as computedtomography or X-rays. An MRI scanner is a device in which the subjectlies within a large, powerful magnet where the magnetic field is used toalign the magnetization of some atomic nuclei in the body, and radiofrequency magnetic fields are applied to systematically alter thealignment of this magnetization. This causes the nuclei to produce arotating magnetic field detectable by the scanner and this informationis recorded to construct an image of a tissue region. Magnetic fieldgradients cause nuclei at different locations to process at differentspeeds, which allows spatial information to be recovered using Fourieranalysis of the measured signal. By using gradients in differentdirections, 2D images or 3D volumes can be obtained in any arbitraryorientation

Various different types of MRI scans can be conducted, includingT₁-weighted MRI, T₂-weighted MRI, and spin density weighted MRI. In someembodiments, the viral imaging agents of the invention are used ascontrast agents to facilitate a T₁-weighted MRI scan. T₁-weighted scansrefer to a set of standard scans that depict differences in thespin-lattice (or T1) relaxation time of various tissues within the body.T₁ weighted images can be acquired using either spin echo orgradient-echo sequences. T₁-weighted contrast can be increased with theapplication of an inversion recovery RF pulse. Gradient-echo basedT₁-weighted sequences can be acquired very rapidly because of theirability to use short inter-pulse repetition times (TR).

Pharmacokinetics and Immune Response to Virus Particles

In some embodiments, administering the rod-shaped plant virus carrier toa subject can generate an immune response. An “immune response” refersto the concerted action of lymphocytes, antigen presenting cells,phagocytic cells, granulocytes, and soluble macromolecules produced bythe above cells or the liver (including antibodies, cytokines, andcomplement) that results in selective damage to, destruction of, orelimination from the human body of cancerous cells, metastatic tumorcells, invading pathogens, cells or tissues infected with pathogens, or,in cases of autoimmunity or pathological inflammation, normal humancells or tissues. Components of an immune response can be detected invitro by various methods that are well known to those of ordinary skillin the art.

Generation of an immune response by the rod-shaped plant virus carrieris typically undesirable. Accordingly, in some embodiments it may bepreferable to modify the rod-shaped plant virus carrier or take othersteps to decrease the immune response. For example, an immunosuppressantcompound can be administered to decrease the immune response. Morepreferably, the rod-shaped plant virus carrier can be modified todecrease its immunogenicity. Examples of methods suitable for decreasingimmunity include attachment of anti-fouling (e.g., zwitterionic)polymers, glycosylation of the virus carrier, and PEGylation.

In some embodiments, the immunogenicity of the rod-shaped plant virus isdecreased by PEGylation to provide a PEGylated rod-shaped plant virus.PEGylation is the process of covalent attachment of polyethylene glycol(PEG) polymer chains to a molecule such as a rod-shaped plant viruscarrier. PEGylation can be achieved by incubation of a reactivederivative of PEG with the rod-shaped plant virus carrier. The covalentattachment of PEG to the rod-shaped plant virus carrier can “mask” theagent from the host's immune system, and reduce production of antibodiesagainst the carrier. PEGylation also may provide other benefits.PEGylation can be used to vary the circulation time of the rod-shapedplant virus carrier. For example, use of PEG 5,000 can provide a viruscarrier with a circulation half-life of about 12.5 minutes, while use ofPEG 20,000 can provide a virus carrier with a circulation half life ofabout 110 minutes.

The first step of PEGylation is providing suitable functionalization ofthe PEG polymer at one or both terminal positions of the polymer. Thechemically active or activated derivatives of the PEG polymer areprepared to attach the PEG to the rod-shaped plant virus carrier. Thereare generally two methods that can be used to carry out PEGylation; asolution phase batch process and an on-column fed-batch process. Thesimple and commonly adopted batch process involves the mixing ofreagents together in a suitable buffer solution, preferably at atemperature between 4 and 6° C., followed by the separation andpurification of the desired product using a chromatographic technique.

A significantly higher dose of rod-shaped plant virus particles remainsin circulation over longer time periods compared to sphericalnanoparticles (SNPs). At 60 minutes post i.v. administration of tobaccomosaic virus (TMV) vs. SNP, 20% injected dose (ID) TMV remained incirculation, while only 5% ID SNPs were detected. This is reflected bythe increased phase II half-lives of 94.9 minutes for TMV and 58.2minutes for SNPs. This phenomenon has been reported using varioussynthetic nanoparticle systems: for example synthetic polymericfilomicelles show enhanced circulation compared to spherical micellesmade of the same polymer, and could be explained by the fact that thehigh aspect ratio materials are less likely taken up by MPS. Geng etal., Nature Nanotechnology 2, 249-255 (2007). There is increasingsupporting data showing that elongated particles avoid clearance byphagocytosis because of their larger and more complex contact anglesbetween the nanoparticle and phagocytotic cell. This has beendemonstrated with gold nanoparticles where the circulation half-life ofrods was significantly longer than their spherical shaped counterpart.Arnida et al., Eur. J. Pharm. Biopharm. 77, 417-423 (2011).

Administration and Formulation of Rod-Shaped Plant Virus Carriers

In some embodiments, the rod-shaped plant virus carrier is administeredtogether with a pharmaceutically acceptable carrier to provide apharmaceutical formulation. Pharmaceutically acceptable carriers enablethe rod-shaped plant virus carrier to be delivered to the subject in aneffective manner while minimizing side effects, and can include avariety of diluents or excipients known to those of ordinary skill inthe art. Formulations include, but are not limited to, those suitablefor oral, rectal, vaginal, topical, nasal, ophthalmic, or parental(including subcutaneous, intramuscular, intraperitoneal, intratumoral,and intravenous) administration. For example, for parenteraladministration, isotonic saline is preferred. For topicaladministration, a cream, including a carrier such as dimethylsulfoxide(DMSO), or other agents typically found in topical creams that do notblock or inhibit activity of the compound, can be used. Other suitablecarriers include, but are not limited to, alcohol, phosphate bufferedsaline, and other balanced salt solutions.

The formulations may be conveniently presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Preferably, such methods include the step of bringing the rod-shapedplant virus carrier into association with a pharmaceutically acceptablecarrier that constitutes one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing theactive agent into association with a liquid carrier, a finely dividedsolid carrier, or both, and then, if necessary, shaping the product intothe desired formulations. The methods of the invention includeadministering to a subject, preferably a mammal, and more preferably ahuman, the composition of the invention in an amount effective toproduce the desired effect. The formulated virus carrier can beadministered as a single dose or in multiple doses.

Useful dosages of the active agents can be determined by comparing theirin vitro activity and the in vivo activity in animal models. Methods forextrapolation of effective dosages in mice, and other animals, to humansare known in the art; for example, see U.S. Pat. No. 4,938,949. Anamount adequate to accomplish therapeutic or prophylactic treatment isdefined as a therapeutically- or prophylactically-effective dose. Inboth prophylactic and therapeutic regimes, agents are usuallyadministered in several dosages until an effect has been achieved.Effective doses of the rod-shaped plant virus carrier vary dependingupon many different factors, including means of administration, targetsite, physiological state of the patient, whether the patient is humanor an animal, other medications administered, and whether treatment isprophylactic or therapeutic.

For administration for targeting or imaging in a mammalian subjectutilizing a rod-shaped plant virus carrier, the dosage of the imagingagent ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5mg/kg, of the host body weight. For example dosages can be 1 mg/kg bodyweight or 10 mg/kg body weight or within the range of 1-10 mg/kg. Asuitable amount of virus particle is used to provide the desired dosage.An exemplary treatment regime entails administration once per every twoweeks or once a month or once every 3 to 6 months. The rod-shaped plantvirus carrier is usually administered on multiple occasions.Alternatively, the rod-shaped plant virus carrier can be administered asa sustained release formulation, in which case less frequentadministration is required. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

The compositions can also include, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes).

For parenteral administration, compositions of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1: Tobacco Mosaic Virus Rods and Spheres asSupramolecular High-Relaxivity MRI Contrast Agents

To compensate for the low sensitivity of magnetic resonance imaging(MRI), nanoparticles have been developed to deliver high payloads ofcontrast agents to sites of disease. The inventors have developedsupramolecular MRI contrast agents using the plant viral nanoparticletobacco mosaic virus (TMV). Rod-shaped TMV nanoparticles measuring300×18 nm were loaded with up to 3,500 or 2,000 chelated paramagneticgadolinium (III) ions selectively at the interior (iGd-TMV) or exterior(eGd-TMV) surface, respectively. Spatial control is achieved throughtargeting either tyrosine or carboxylic acid side chains on the solventexposed exterior or interior TMV surface. The ionic T₁ relaxivity per Gdion (at 60 MHz) increases from 4.9 mM⁻¹s⁻¹ for free Gd(DOTA) to 18.4mM⁻¹s⁻¹ for eGd-TMV and 10.7 mM⁻¹s⁻¹ for iGd-TMV. This equates to T₁values of ˜30,000 mM⁻¹s⁻¹ and ˜35,000 mM⁻¹s⁻¹ per eGd-TMV and iGd-TMVnanoparticle. Further, the inventors show that interior-labeled TMV rodscan undergo thermal transition to form 170 nm-sized sphericalnanoparticles containing ˜25,000 Gd chelates and a per particlerelaxivity of almost 400,000 mM⁻¹s⁻¹ (15.2 mM⁻¹s⁻¹ per Gd). This worklays the foundation for the use of TMV as a contrast agent for MRI.

EXPERIMENTAL

TMV propagation: TMV was propagated in N. benthamiana plants. TMV wasextracted in yields of 4.5 mg of virus per gram infected leaf materialusing established extraction methods. Boedtker, H, Simmons, N, JACS, 80,2550-2556 (1958). Virus concentration in plant extracts was determinedby UV-Vis absorbance (ε_(260 nm)=3.0 mg⁻¹ mL cm⁻¹), and virus integritywas determined by size exclusion chromatography (SEC), and transmissionand scanning electron microscopy (TEM and SEM) imaging.

TMV bioconjugation: To decorate the exterior TMV surface, the phenolring of tyrosine underwent an electrophilic substitution (pH=9, 30 min.)with the diazonium salt generated from 3-ethynylaniline (25 molarequivalents (eq)) to incorporate a terminal alkyne. The resultingnanorods are designated eAlk-TMV. Bruckman et al., ChemBioChem 9,519-523 (2008). Similarly, a terminal alkyne was incorporated onto theinterior channel of TMV by targeting glutamic acid residues, designatediAlk-TMV. Wu et al., Journal of Materials Chemistry 21, 8550-8557(2011). This was achieved by mixing propargyl amine (25 eq) with EDC(ethyldimethylpropylcarbodiimide, 45 eq) and HOBt(n-hydroxybenzotriazole, 45 eq) for 24 hours. The HOBt is used tosuppress EDC side product formation. Following sucrose gradientultracentrifugation purification, the structural integrity of theparticles was confirmed with TEM and SEC and the labeling efficiency wasconfirmed with MALDI-TOF MS.

Efficient conjugation of Gd(DOTA) azide to terminal alkyne labeled TMV(eAlk- and iAlk-TMV) is accomplished via a copper-catalyzed azide-alkynecycloaddition (CuAAC) to form exterior or interior Gd conjugated TMV,designated eGd-TMV and iGd-TMV, respectively (FIG. 3 ). Initially, GdCl₃was incubated with commercially available (azido-monoamide-1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid;Macrocyclics) DOTA azide while maintaining a pH ˜6-7 (adjustedperiodically with NaOH) over a six day-period to produce Gd(DOTA) azide.For both interior and exterior conjugation, the same protocol was used.Briefly, alkyne-labeled TMV (2 mg/ml) in 0.1 M potassium phosphatebuffer pH 7.0 was mixed with Gd(DOTA) azide (5 eq to CP), aminoguanidine(2 mM), ascorbic acid (2 mM), and copper sulfate (1 mM) for 15 minutes.The reaction mix was purified using a 10-40% sucrose gradient andultracentrifugation, and analyzed by TEM, SEM, SEC, and MALDI-TOF MS.

Thermal transition to SNPs: The standard protocol for thermal transitionof native TMV rods to SNPs is heating of the sample at 0.1 mg/mL for 10seconds at 96° C. with a Peltier thermal cycler. Alternatively, foriGd-TMV, PEG 8 kDa (0.5% w/v) was added to the reaction mix andincubation time was increased to 15 seconds.

MALDI-MS analysis: For MALDI-MS analysis, native and modified TMV (weredenatured using guanidine hydrochloride (6 μL, 6 M) to the sample at10-20 μg in 24 μL 0.1 M potassium phosphate buffer and mixing for 5 minat room temperature. Denatured proteins were spotted on MTP 384 massivetarget plate using Zip-Tips_(μC18) (Millipore). MALDI-MS analysis wasperformed using a Bruker Ultra-Flex I TOF/TOF mass spectrometer.

Size exclusion chromatography (SEC): All labeled particles were analyzedby SEC using a Superose6 column on the ÄKTA Explorer chromatographysystem (GE Healthcare). Samples (100 μg/100 μL) were analyzed at a flowrate of 0.5 mL/min using 0.1 M potassium phosphate buffer (pH 7.0).

Transmission electron microscopy (TEM): Drops of TMV rods or SNPs in DIwater were placed on copper TEM grids (5 μL, 0.1 mg/mL), allowed toadsorb for 5 minutes, washed with DI water, and negatively stained with2% (w/v) uranyl acetate for 2 minute. Samples were examined using aZeiss Libra® 200FE transmission electron microscope operated at 200 kV.

Gel electrophoresis: Denaturing gel electrophoresis was used to analyzeprotein subunits, specifically proteins were analyzed on denaturing4-12% NuPAGE gels (Invitrogen) using 1×MOPS running buffer (Invitrogen)and 10 μg of sample. After separation, the gel was photographed using anAlphaImager (Biosciences) imaging system after staining with CoomassieBlue. ImageJ software was used for band analysis and to determine theprotein concentration per SNP.

Scanning electron microscopy (SEM): Samples were dried onto glass coverslips and then mounted on the surface of an aluminium pin stub with useof double-sided adhesive carbon discs (Agar Scientific). The stubs werethen sputter-coated with gold in a high-resolution sputter coater (AgarScientific, Ltd.) and transferred to a Hitachi 4500 scanning electronmicroscope.

ICP-OES measurements: The Gd per VNP ratio was determined using anICP-OES (Perkin-Elmer ICP-OES 3300 DV) located in the Geology Departmentat Kent State University.

Relaxivity measurements: The ionic relaxivity of the engineered VNPs wastested using a pre-clinical 7.0 T (300 MHz) MRI (Bruker BioSpec®70/30USR), a clinical 1.5 T (64 MHz) MRI (Siemens Espree), and a BrukerMinispec® mq60 relaxometer (60 MHz). A standard inversion recoverysequence protocol was used to determine the T₁ values on each of theinstruments.

Results and Discussion

Spatially-Controlled Loading of MR Contrast Agents to the Exterior andInterior Surface of TMV.

TMV was propagated in N. benthamiana plants. Pure TMV nanorods wereextracted in yields of 4.5 mg of virus per gram infected leaf materialusing established extraction methods. The exterior and interior surfacesof TMV's hollow rod can be efficiently functionalized using previouslyestablished bioconjugation protocols. FIG. 2 shows the high-resolutioncrystal structure of TMV highlighting carboxylic acids and tyrosine sidechains (PDB ID 2TMV). Previous studies have indicated that exteriortyrosine 139 residues and interior glutamic acid 97 and 106 residues canbe modified and functionalized using diazonium coupling orcarbodiimide-based conjugation reactions.

The inventors explored both the exterior and interior surface forlabeling with chelated gadolinium compounds. Using the interior surfaceprovides the advantage that the exterior surface remains available forfurther tailoring with tissue-specific ligands. On the other hand,attaching the contrast agent to the exterior surface provides anopportunity to load the interior with drugs. To decorate the exteriorTMV surface, tyrosine residues were targeted with the diazonium saltgenerated from 3-ethynylaniline to yield eAlk-TMV. Bruckman et al.,ChemBioChem 9, 519-523 (2008). Similarly, a terminal alkyne wasincorporated onto the interior channel of TMV by targeting glutamic acidresidues, designated iAlk-TMV. Wu et al., Journal of Materials Chemistry21, 8550-8557 (2011). Following sucrose gradient ultracentrifugationpurification, the structural integrity of the particles was confirmedwith TEM and SEC and the labeling efficiency was confirmed withMALDI-TOF MS.

Efficient conjugation of Gd(DOTA) azide to terminal alkyne labeled TMV(eAlk- and iAlk-TMV) was accomplished via a copper-catalyzedazide-alkyne cycloaddition (CuAAC) to form exterior or interior Gdconjugated TMV, designated eGd-TMV and iGd-TMV, respectively (FIG. 3 ).eGd-TMV and iGd-TMV formulations were purified using a 10-40% sucrosegradient and ultracentrifugation. This reaction gave an overall yield of50-60% i/eGd-TMV, as confirmed by UV-Vis absorption to measure the TMVconcentration (Abs_(260 nm)=3 for 1 mg/ml) and SDS-PAGE. The structuralintegrity of the modified TMV particles was confirmed with sucrosegradients (matching light scattering region to native TMV), SEC, andTEM. A representative TEM image of iGd-TMV is shown in FIG. 4B. Thesuccessful incorporation of Gd-DOTA-azide onto the interior or exteriorsurfaces of TMV was confirmed using MALDI-TOF MS (FIGS. 3C and 3D) andICP-OES (see Table 1).

TABLE 1 Longitudinal relaxivity values for Gd-TMV particles. Relaxivityper Gd (and per particle) mM⁻¹s⁻¹ VNP Gd/VNP 60 MHz 64 MHz 300 MHzeGd-TMV 1,712 18.4 (31,501) 15.7 (26,896) 6.7 (11,402) iGd-TMV 3,41710.7 (36,562) 11.0 (37,519) 4.7 (15,932) iGd-SNP 25,815  15.2 (392,388) 13.2 (340,758) 3.7 (95,515) Gd(DOTA) 1 4.9 4.9 4.9

For eGd-TMV, the mass spectrum (MS) shown in FIG. 3C displays peaksattributed to eAlk-TMV/wt-TMV coat proteins (CP) (eAlk, 17713 m/z), CPswith one (1-Gd, 18339 m/z) and two (2-Gd, 19094 m/z) Gd(DOTA) moleculesattached. Similarly, the MS of iGd-TMV shown in FIG. 3D displays peaksattributed to iAlk-TMV/wt-TMV CPs (iAlk, 17618 m/z), CPs with one (1-Gd,18359 m/z), two (2-Gd, 19044 m/z), and three (3-Gd, 19671 m/z) Gd(DOTA)molecules attached. The differences in mass values obtained areattributed to the DOTA molecule only, indicating that the chelated Gdions did not remain chelated to DOTA after ionization. It should benoted that the presence of Gd was confirmed using ICP-OES.

Interestingly, MALDI-TOF MS characterizations of both the exterior andinterior labeling indicate one additional amino acid modification per CPthan previously reported. Schlick et al., JACS 127, 3718-3723 (2005).For exterior conjugation, the MS indicates a majority of the CPs thatmake up eGd-TMV have one Gd(DOTA), likely attached to TYR139, a smallamount of CPs that are un-labeled, and a small amount of CPs thatcontain two Gd(DOTA) molecules per CP. Of the four tyrosine residuescontained in the TMV coat protein, TYR139, TYR2, TYR70, and TYR72 (seeFIG. 2 ), only Tyr139 has proven to be the primary reactive tyrosine.Based on the crystal structure it appears that TYR2 is solvent-exposed(more than TYR70 and TYR72), and the inventors believe that TYR2 is thepotential second attachment site (see FIG. 2 ). For interior labeling,glutamic acids GLU97 and GLU106 have been proven to undergobioconjugation while the third modification site remains unclear. TheTMV coat protein includes several aspartic and glutamic acids that couldserve as potential attachment sites (highlighted in blue in FIG. 2 ).

Quantitative labeling of TMV's interior and exterior surface with Gd wasconfirmed using ICP-OES. Data indicate that the exterior (eGd-TMV) wasloaded with 1,712 Gd per particle and the interior (iGd-TMV) was loadedwith 3,417 Gd per particle. The ICP-OES results are in agreement withthe MALDI-TOF MS results. The results are exciting at least in partbecause previous studies have not indicated labeling of a secondtyrosine or third carboxylic acid.

Thermal Transition of Contrast Agent Loaded TMV Rods into SNPs

In view the capability of TMV to form uniform SNPs (FIG. 4A), theinventors explored the thermal transition of chemically modified TMVparticles (FIG. 4 ). Bruckman et al., J Mater Chem B Mater Biol Med.Mar. 14; 1, 1482-1490 (2013). Initially, the exterior modified TMVparticles (eGd-TMV) were tested. It was found that no SNPs were formedafter heating for 10 seconds at 96° C. (FIG. 4C). The conditions wereexpanded to extended incubation times (up to 30 seconds) and addition ofadditives such as PEG, urea, guanidinium chloride, triton X-100 and athigh and low ionic strengths and found that no SNPs were formed.Finally, the inventors attempted to form SNPs with eAlk-TMV, thinkingthe DOTA group was too large and blocking the assembly. Again the SNPsdid not form and only broken protein aggregates were found. This maysuggest that TYR139 might play a role in the formation and stability ofSNPs.

Subsequently, the thermal transition of interior modified TMV (iGd-TMV)to SNPs was tested. Using the standard protocol, i.e. heating for 10seconds at 96° C. with a Peltier thermal cycler, formation of SNPs wasnot noticeable. While a lack of rod-shaped particles indicated that TMVwas denatured, only irregular protein aggregates were observed.Similarly, a variety of additives were used to either increase ordecrease TMVs stability (listed above). It was found that addition ofPEG 8 kDa (0.5% w/v) to the reaction mix improved the stability of SNPsand decrease non-specific protein aggregation. After heating for 10seconds, more regular SNP formation was found, however, the transitionwas incomplete, meaning that many rod-shaped TMV particles were stilldetectable in the sample (FIG. 4D). The rods appeared to be feeding intothe SNPs indicating an end-in feeding/melting mechanism. Next, theincubation time was increased from 10 to 15 seconds and found that allof the rods were transitioned to SNPs, as seen with TEM (FIG. 4E) andSEM (FIG. 4F). A longer heating time is required to fully transitioniGd-TMV into iGd-SNPs compared to native TMV. The inventors propose thatthe requirement of additional incubation time is because the interiormodified TMV are more stable than native TMV. More stable interiormodified TMV particles were confirmed using a differential scanningcalorimeter. Native TMV was found to fall apart at 65° C., whereasiAlk-TMV remained stable until a temperature of 80° C. was reached.Modification of glutamic acids 97 and 106 has been shown to decrease theelectrostatic repulsion between CPs, therefore leading to strongerattraction between CPs. Lu et al., Virology 225, 11-20 (1996).

Analysis of the SNP size distribution was done using TEM (FIG. 4E), SEM(FIG. 4F) and dynamic light scattering (DLS) with a Nanosight sizeanalyzer (FIG. 4G) and a standard DLS instrument (Brookhaven). It wasfound that there is some variability from experiment to experiment withthe size of the SNP batches varying between 150 nm to 200 nm. Within aparticular batch, however, there is a narrow size distribution. The SNPbatch utilized for the described studies, measured a hydrodynamic radiusof 170±41 nm in diameter as determined by Nanosight (FIG. 4G) and DLS.This is in agreement with TEM and SEM measurements that indicated a SNPsize of 152 nm±58 nm (the smaller size is explained that in SEM and TEMdried samples are measured whereas Nanosight and DLS record thehydrodynamic radii).

The Gd loading per SNP was determined using a combination of ICP-OES forGd concentration and SDS-PAGE for protein concentration. Here, when itis assumed that the coat proteins form densely packed SNPs upon thermaltransition, a 170 nm-sized SNP would contain ˜75,400 coat proteins (35.4times the number of coat protein found in a single TMV rod). The proteinconcentration was estimated using SDS-PAGE protein gel electrophoresisfollowed by Coomassie staining and band analysis using ImageJ software.Based on the SDS-PAGE and size analysis, the inventors estimated that a1 mg/ml solution of SNPs contained 4.73×10¹¹ particles/mL, or a molarconcentration of 7.05×10⁻¹⁰ M. ICP-OES analysis of the same 1 mg/mlsolution of SNPs contained 2.85 ppm Gd, or 1.82×10⁻⁵M, yielding SNPswith 25,815 Gd per SNP (see Table 1).

The ionic relaxivity of the engineered VNPs was tested using apre-clinical 7.0 T (300 MHz) MRI (Bruker BioSpec® 70/30USR), a clinical1.5 T (64 MHz) MRI (Siemens Espree), and a Bruker Minispec® mq60relaxometer (60 MHz). A standard inversion recovery sequence protocolwas used to determine the T₁ values on each of the instruments. Shown inFIG. 5A is the inversion recovery image (T₁=2000 ms) of iGd-TMV andeGd-TMV phantoms taken on the clinical MRI (64 MHz, 1.5 T). Theconcentrations increase from left to right with the phantom on far leftbeing water. In order to determine the ionic relaxivities of Gd, 1/T₁(units=1/seconds) was plotted against the concentration of Gd (in μM)for each formulation and field strength; the slope of each correlationline is the ionic relaxivity (r₁, see FIG. 5B+C). The relaxivity of theentire particle was computed by multiplying the ionic relaxivity by thenumber Gd ions per particle determined by ICP-OES. Similar analysis wasperformed on the Gd-SNPs (data are summarized in Table 1). Next, ionicrelaxivity values were determined using a pre-clinical MRI andrelaxometer with a similar inversion recovery sequence (see Table 1).The ionic relaxivity increased at lower field strengths. Caravan et al.,Contrast Media & Molecular Imaging 4, 89-100 (2009).

As with other macromolecular carriers, the relaxivity of DOTA chelatedGd ions was found to increase after conjugation to TMV, compared to freeGd(DOTA) in solution. The increase in ionic relaxivity is greater forexterior labeling of TMV compared to the interior labeling, 18.4 mM⁻¹s⁻¹and 10.7 mM⁻¹s⁻¹, respectively. This is primarily attributed to thedifference in molecular attachment site. Exterior modification iscarried out targeting tyrosine side chains and interior loading isaccomplished through modification of glutamic acids. The ring structureof the tyrosine side chain induces rigidity, whereas the alkyl chain inthe glutamic acids is comparatively flexible. The more rigid theattachment site, the higher the enhancement in relaxivity. This isconsistent with previous reports that showed that amino acid stiffnesslowers the tumbling rate thus increasing relaxivity. For example,exterior lysine residues of MS2 were labeled with bis(HOPO) ligands tochelate Gd, they exhibited an ionic T₁ relaxivity of 23.2 mM⁻¹s⁻¹, whileinterior tyrosine residues labeled with the same bis(HOPO) liganddemonstrated an ionic T₁ relaxivity of 31.0 mM⁻¹s⁻¹ (at 60 MHz). Hookeret al., Nano Letters 7, 2207-2210 (2007).

Additionally, after transition of iGd-TMV to SNPs, the ionic relaxivityincreases from 10.7 mM⁻¹s⁻¹ to 15.2 mM⁻¹s⁻¹ at 60 MHz. One potentialexplanation for the increase is because after transition to SNPs, theoverall mobility (correlated to the tumbling rate) of each Gd may belowered because of molecular crowding from the dense packing of proteinsinto spheres. Here, it is important to note the drastic difference inper nanoparticle relaxivity between TMV rods and spheres. Based on ICPresults, the iGd-TMV particles have 3,417 Gd atoms per rod, which givesa per particle relaxivity of 36,562 mM⁻¹s⁻¹, while the Gd-SNPs contain25,815 Gd atoms per sphere giving a per particle relaxivity of 392,388mM⁻¹s⁻¹.

Based on their biocompatibility, monodispersity and ability to undergomultiple rounds of site-selective chemical and/or genetic modification,several icosahedral VNPs have previously been utilized as scaffolds forthe presentation of MRI contrast agents, these include cowpea chloroticmottle virus (CCMV), cowpea mosaic virus (CPMV), and bacteriophages MS2and Qβ. In all cases enhancements of ionic T1 relaxivities aboveFDA-approved Magnevist® (data are summarized in FIG. 6 ). The Gd-TMVparticles generated in this study show similar T1 relaxivityenhancements to VNPs decorated with Gd chelated with DOTA or DTPAligands, see FIG. 6 . While the ionic relaxivity of TMV is comparable toother magnetic VNPs, the rod-shaped Gd loaded TMV particles have a fourtimes higher per particle relaxivity (of more than 30,000 mM⁻¹s⁻¹)compared to icosahedral VNPs (see FIG. 6 ). These results are excitingand expected because while the size of TMV is bigger (volume=7.6×10⁴ nm³of TMV vs 1.4×10⁴ nm³ for a 30 nm-sized icosahedron), the relaxivity pervolume ratio (R1/V) is similar. The R1/V for eGd-TMV and iGd-TMV is 0.41and 0.48, respectively, while the R1/V for the spherical VNPs rangesfrom 0.13 to 0.52. To date the highest per Gd relaxivities were reportedby the Francis Lab, who utilized a special HOPO ligand. Raymond et al.,Bioconjugate Chemistry 16, 3-8 (2005).

Finally, the enhancement in ionic relaxivity per Gd is maintained afterthermal transition to SNPs. Phantom MRI tests indicate that therelaxivity is even further enhanced. The per particle relaxivity of theSNPs (4×10⁵ mM⁻¹s⁻¹) bridges the gap between contemporary VNPs (T1relaxivity near 10⁴ mM⁻¹s⁻¹) and dendrimers, silica nanoparticles andperfluorocarbons, which have per particle T1 relaxivities in the 10⁶mM⁻¹s⁻¹ range.

CONCLUSION

In conclusion, the inventors have developed a plant viral-basednanoparticle platform suitable for application as an imaging agent.Covalent attachment of chelated gadolinium ions to the supramolecularcarrier leads to enhanced ionic relaxivity of the Gd ions based onreduced tumbling rates. Multivalent display leads to relaxivity pernanoparticle four times higher than over VNP contrast agents.Furthermore, the transition of rod-shaped TMV to SNPs improved the ionicT1 relaxivity per Gd based on molecular crowding, while furtherincreasing the loading of Gd per particle yielding a protein based MRIcontrast agent with a T1 relaxivity of 400,000 mM⁻¹s⁻¹. The Gd-loadedTMV rods and spheres reach T1 relaxivities comparable tostate-of-the-art dendrimers.

Example 2: Dual-Modal MRI and Fluorescence Imaging of AtheroscleroticPlaques In Vivo Using VCAM Targeted Tobacco Mosaic Virus

The nanoparticles formed by plant viruses are emerging tools formolecular imaging in medicine. Here, the rod-shaped tobacco mosaic viruswas used to target and image atherosclerotic plaques in vivo. TMV wasloaded with magnetic resonance (MR) and fluorescence contrast agents toprovide a dual-modal imaging platform. Targeting to atheroscleroticplaques was achieved with vascular cell adhesion molecule (VCAM)receptors present on activated endothelial cells. Dual, molecularimaging was confirmed using a mouse model of atherosclerosis.

Methods

Isolation of TMV. TMV particles were isolated from Nicotiana benthamianaor N. rusitca plants using a previously established protocol. BoedtkerH, Simmons N S. JACS, 80:2550-6 (1958). The TMV concentration wasdetermined based on UV-Vis absorbance at 260 nm with an extinctioncoefficient of 3.0 mL mg⁻¹ cm⁻¹.

Bioconjugation of TMV particles. A peptide was chosen to target vascularcell adhesion molecule (VCAM-1) receptors on the surface of activatedendothelial cells. Nahrendorf et al., Circulation. 114:1504-11 (2006)The VCAM-1 targeting peptide labeled with an azide group was synthesizedusing standard peptide synthesis techniques. Targeted (VCAM-TMV) andnon-targeted (PEG-TMV) rods were synthesized using the followingsequence of established reactions. Bruckman et al., J Mater Chem B MaterBiol Med. 1:1482-90 (2013). First, the exterior was labeled with aterminal alkyne by targeting tyrosine 139 residues. Azido PEG or VCAMwas attached to the exterior alkyne using a copper-catalyzedazide-alkyne cycloaddition (CuAAC) reaction. Next, the interior waslabeled with terminal alkynes by targeting glutamic acids 97 and 106with a primary amine and EDC. Finally, MRI contrast agents (Gd(DOTA)azide) and fluorescent molecules (sulfo-Cy5 azide) were attached to TMVusing the CuAAC reaction. Modified TMV particles were characterized forlabeling efficiency, structural integrity, and magnetic T₁ relaxivityenhancement. Labeling efficiency was confirmed using MALDI-TOF massspectrometry, SDS-PAGE electrophoresis, ICP-OES (for Gd), and UV-Visabsorbance (for Cy5). Particle integrity was confirmed by TEM and SEC.The ionic relaxivity of engineered Gd-loaded TMV particles was testedusing a Bruker Minispec® mq60 relaxometer.

MALDI-MS analysis. For MALDI-MS analysis, native and modified TMV weredenatured using guanidine hydrochloride (6 μL, 6 M) to the sample at10-20 μg in 24 μL 0.1 M potassium phosphate buffer and mixing for 5 minat room temperature. Denatured proteins were spotted on MTP 384 massivetarget plate using Zip-Tips_(μC18) (Millipore). MALDI-MS analysis wasperformed using a Bruker Ultra-Flex I TOF/TOF mass spectrometer.

Size exclusion chromatography (SEC). All labeled particles were analyzedby SEC using a Superose6 column on the AKTA Explorer chromatographysystem (GE Healthcare). Samples (100 μg/100 μL) were analyzed at a flowrate of 0.5 mL/min using 0.1 M potassium phosphate buffer (pH 7.0).

Transmission electron microscopy (TEM). Drops of TMV formulations in DIwater were placed on copper TEM grids (5 μL, 0.1 mg/mL), allowed toadsorb for 5 minutes, washed with DI water, and negatively stained with2% (w/v) uranyl acetate for 2 minute. Samples were examined using aZeiss Libra® 2001-th transmission electron microscope operated at 200kV.

Gel electrophoresis. Denaturing gel electrophoresis was used to analyzeprotein subunits, specifically proteins were analyzed on denaturing4-12% NuPAGE gels (Invitrogen) using 1×MOPS running buffer (Invitrogen)and 10 μg of sample. After separation, the gel was photographed using anAlphalmager (Biosciences) imaging system after staining with CoomassieBlue. ImageJ software was used for band analysis and to determine thedegree of labeling with PEG and VCAM.

Relaxivity measurements. The ionic relaxivity of the Gd(DOTA)-modifiedTMV was tested using a Bruker Minispec mq60 relaxometer (60 MHz). The Gdconcentration was determined using an ICP-OES. Multiple concentrationsof TMV were used with a standard inversion recovery sequence protocol todetermine the T₁ values.

Animal protocols. All experiments were carried out using IACUC approvedprocedures. ApoE^(−/−) mice were used for all experiments. ApoE^(−/−)mice had an average age of 22 weeks were fed a western diet (1.25%cholesterol, 20% fat, Research Diets Inc.) for 14-18 weeks were injectedvia the tail vein with virus particles at an amount of 10 mg/kg. HealthyC57BL/6 mice of the same age served as negative controls.

Ex vivo fluorescence imaging. Mice were euthanized and dissected toremove aortas 3 hours post administration of TMV and respectivecontrols. The aorta was then fixed in 4% (v/v) paraformaldehyde in 30%(v/v) sucrose overnight at 4° C. After fixation, the aorta was cleanedto remove fatty connective tissue. The cleaned aorta was then imagedusing a Maestro fluorescence imaging system to detect Cy5 fluorescencesignal. Image cubes were obtained with an exposure time of 800 ms perstep. The obtained image cubes were background subtracted prior toquantitative image analysis.

Immunofluorescence Imaging. Immediately after ex vivo fluorescenceimaging, the cleaned aortas were cut into 10-12 2-4 mm long sections andembedded in OCT and flash frozen. The frozen samples were cryosectionedto 10 μm sections and mounted on Fisherbrand ColorFrost Plus microscopeglass slides for staining. Freshly sectioned aortas were stained formacrophage cell marker CD68 (BioLegend) and mounted using mounting mediacontaining DAPI (Fluoroshield with DAPI, Sigma).

MRI analysis. In vivo MRI scans were performed using a Bruker BioSpin®7.0 T 70/30USR MRI system. This system has been outfitted with an RFmouse coil. Mice were anesthetized for all procedures (isoflurane 1.5%;O₂ 2.5 L/min) and their respiration, body temperature, and heart rate(ECG) were monitored real-time. Following multiple scouting scans, aT₁-weighted Multi Slice Multi Echo (MSME) black-blood fat-suppressionsequence was optimized to detect the aorta wall with the followingparameters: TR/TE=600/8.0 ms, 8 axial slices 1 mm thickness with 1.5 mmslice separation, two averages, matrix=256×256, field of view=2.98 cm,acquisition time=10:14 minutes. Respiration and ECG triggering wasapplied per slice. Images were taken prior and post TMV administration,formulations were injected while the animal remained in the MRI machine.Sequential scans were performed for up to 150 minutes. Statisticalanalysis of MRI results was performed using MatLab program to determinethe contrast to noise ratio (CNR).

Results

Bioconjugation to synthesize VCAM-TMV and PEG-TMV. TMV particles weremodified with contrast agents, PEG and targeting ligands to targetVCAM-1 receptors that are overexpressed on endothelial cells in areas ofatherosclerotic plaque development (FIG. 7 ). The inventors chose knownVCAM-1 ligand: VHPKQHR. VCAM-1 peptide and PEG were synthesized toincorporate a linker with an azide functional group for conjugation toTMV using CuAAC chemistry. A PEG₂₀₀₀ labeled TMV was synthesized as anon-targeting nanoparticle control. The specific sequence for modifyingTMV is outlined in FIG. 7 . In brief, first the exterior surface ismodified with alkyne ligation handles followed by modification with PEGand VCAM peptide. Second, the interior is modified with alkyne ligationhandles followed by modification with optical and MR contrast agents (Cyand Gd(DOTA)). TEM imaging and SEC analysis indicate that the particlesremain structurally sound; further SEC indicates covalent modificationwith Cy5 (as indicated by co-elution of the dye-specific peak at 650 nmwith the protein peak at 260 nm, see FIG. 8 ). A combination ofSDS-PAGE, MALDI-TOF, UV-Vis, and ICP-OES measurements were performed todetermine the degree of labeling: MADLI-TOF results are in goodagreement with sequential modification of the TMV coat proteins.Further, based on SDS-PAGE lane analysis (FIG. 8 , ImageJ), theinventors estimate coverage with ˜500 VCAM peptides and PEG per VCAM-TMVand PEG-TMV, respectively, which corresponds to 25% of the coat proteinsbeing labeled (TMV consists of 2130 identical coat protein units).UV-Vis was used to determine the degree of Cy5 labeling and ICP-OES wasused to determine the number of incorporated Gd(DOTA). It was found thatVCAM-TMV and PEG-TMV were labeled with ˜460 and ˜510 Cy5 dyes,respectively, thus also covering 25% of the available coat proteins. Thedegree of labeling was significantly higher—a desired results, becausethe higher the density of the chelated Gd(DOTA) ions, the higher the T₁relaxivity per nanoparticle; VCAM-TMV was loaded with ˜1,200 chelated Gdions, resulting in a per particle relaxivity of 17,567 mM^(−l)s⁻¹ at 60MHz. (FIG. 9 ).

VCAM-TMV targeting was confirmed using ex vivo fluorescence imaging ofaortas. ApoE^(−/−) mice were able to develop sufficient plaque coveragein 14-18 weeks on a high fat/cholesterol diet (based on posthistological analysis). Along with VCAM-TMV (n=4) for the targetingcontrast agent and PEG-TMV (n=3) as the negative nanoparticle control,PBS (n=3) and free contrast agent (sulfo-Cy5-azide and Gd(DOTA)-azide)(n=1) were injected at concentrations matching the TMV labeled injectiondose. Finally, VCAM-TMV was injected into the age-matched healthy mousemodel C57BL/6 (n=1) to demonstrate that VCAM-TMV particles do notaccumulate at healthy endothelial cells and arteries. Data indicateselective targeting and accumulation of TMV-VCAM in aortas fromApoE^(−/−) fed on a high western diet. Fluorescence intensity fromaortas from animals injected with VCAM-TMV (fluorescence intensity(FI)=19.6) was significantly higher than signals obtained from PEG-TMV(FI=7.2), PBS (FI=4.9), and the aorta from a C57BL/6 mouse (FI=2.4).

Immunofluorescense imaging of cryo-sectioned aortas further confirmedVCAM-TMV targeting and accumulation in areas of atherosclerotic plaque.Cryosections of aortas were stained for macrophages (CD68 antibody) toconfirm the presence of plaques. Images of an aorta section from miceinjected with VCAM-TMV, PEG-TMV, and PBS were obtained. Each mouse aortawas quantitatively analyzed for the number of sections that containedplaques and that also contained fluorescent TMV signal and found that70% of plaque sections from aortas injected with targeted VCAM-TMVparticles (n=4) showed TMV accumulation, while only 18% of aortasections that were positive for PEG-TMV signals. No TMV (Cy5) signal wasdetected in aorta sections that did not indicate any areas of plaquedevelopment, therefore indicating that TMV does no non-specificallyadhere to the vessel wall. VCAM-TMV was localized at the surface of theplaque, indicating targeting of activated endothelial cells expressingVCAM. There was no indication that VCAM-TMV particles were taken up bymacrophages and incorporated into plaques.

MR imaging atherosclerotic plaques in ApoE^(−/−) mice usingmolecularly-targeted VCAM-TMV. MR imaging of ApoE^(−/−) mice wasconducted using the Gd(DOTA)-labeled VCAM-TMV sensors. Significantincrease in contrast-to-noise (CNR) was observed in the vessel wall ofthe abdominal aorta (FIG. 10A). The increase in CNR increased over timeand leveled off at around 60 minutes. Not all slices indicatedaccumulation of paramagnetic contrast agent, which is in agreement withnot all slices containing plaques (this is also consistent withfluorescence analysis, see above). Free Gd(DOTA)) and PBS did not showany increase in CNR in the vessel wall (FIGS. 10B and C, respectively).

In summary, the data support the successful development of amolecularly-targeted TMV-based probe for dual MR and optical imaging ofatherosclerotic plaques in mice.

The complete disclosure of all patents, patent applications, andpublications, and electronically available materials cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. In particular,the inventors are not bound by theories described herein. The inventionis not limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

What is claimed is:
 1. A method of generating an image of a tissueregion of a subject, the method comprising: administering to the subjecta diagnostically effective amount of a spherical nanoparticle imagingplatform, the spherical nanoparticle imaging platform including aspherical arrangement of the coat proteins of one or more rod-shapedplant virus particles linked to an imaging agent on an interior surfaceof the virus particle, formed by thermal transition of the rod-shapedvirus particles, wherein the imaging platform has a longitudinalrelaxivity of greater than about 10 mM⁻¹S⁻¹ per linked imaging agentwhen measured at 60 MHz at a physiological pH; and generating an imageof the tissue region of the subject to which the rod-shaped plant virusparticle has been distributed.
 2. The method of claim 1, wherein therod-shaped plant virus is a tobacco mosaic virus.
 3. The method of claim1, wherein the method of generating an image is magnetic resonanceimaging, and the imaging agent is a chelated lanthanide.
 4. The methodof claim 1, wherein the spherical nanoparticle imaging platform isadministered together with a pharmaceutically acceptable carrier.
 5. Themethod of claim 1, wherein the tissue region includes a tumor.
 6. Themethod of claim 1, wherein the tissue region includes a blood vessel. 7.The method of claim 1, wherein the imaging platform has a longitudinalrelaxivity of greater than about 15 mM⁻¹S⁻¹ per linked imaging agentwhen measured at 60 MHz at a physiological pH.
 8. A method of generatingdual modal imaging of an atherosclerotic plaque in a blood vessel of asubject, the method comprising: administering to the subject adiagnostically effective amount of a rod-shaped plant virus particle,the rod-shaped plant virus particle having a magnetic resonance imagingagent and a fluorescent imaging agent linked to an interior surface ofthe virus particle and a vascular cell adhesion molecule (VCAM)targeting ligand linked to the exterior surface of the virus particle;and generating a magnetic resonance image and a fluorescent opticalimage of the blood vessel of the subject to which the rod-shaped plantvirus particle has been distributed.
 9. The method of claim 8, whereinthe rod-shaped plant virus is a tobacco mosaic virus.
 10. The method ofclaim 8, wherein the rod-shaped plant virus particle is administeredtogether with a pharmaceutically acceptable carrier.
 11. The method ofclaim 8, wherein the magnetic resonance imaging agent is a chelatedlanthanide.
 12. The method of claim 11, wherein the lanthanide isgadolinium.
 13. The method of claim 8, wherein the fluorescent imagingagent is a cyanine 5 (Cy5) dye.
 14. The method of claim 8, wherein theVCAM targeting ligand is a VCAM-1 ligand having the amino acid sequenceSEQ ID NO: 1 (VHPKQHR).
 15. A method of generating a sphericalnanoparticle imaging platform, the method comprising: providing areaction mixture, the reaction mixture including TMV plant virus rods ata concentration of 0.1 mg mL⁻¹ in H2O and polyethylene glycol (PEG) 8kDa at 0.5% w/v, the TMV plant rods having an interior surface and anexterior surface and at least one imaging agent that is linked to theinterior surface of the TMV plant virus; and heating the reactionmixture for about 15 seconds at about 96° C., wherein heating results inthe thermal transition of the TMV plant rods into spherical nanospheres(SNPs).
 16. The method of claim 15, wherein the imaging platformproduced has a longitudinal relaxivity of greater than about 10 mM⁻¹S⁻¹per linked imaging agent when measured at 60 MHz at a physiological pH.17. The method of claim 15, wherein the imaging platform produced has alongitudinal relaxivity of greater than about 15 mM⁻¹S⁻¹ per linkedimaging agent when measured at 60 MHz at a physiological pH.
 18. Themethod of claim 15, wherein the imaging agent is a magnetic resonanceimaging agent.
 19. The method of claim 15, wherein the magneticresonance imaging agent is a chelated lanthanide.
 20. The method ofclaim 19, wherein the lanthanide is gadolinium.